Role And Mechanisms Of 20-HETE On AngⅡ-induced Cardiac Hypertrophy

Cardiac hypertrophy is a common pathological and physiological process of many cardiovascular diseases.It is an independent risk factor for cardiovascular diseases such as arrhythmia and myocardial infarction.Angiotensin II（AngII）,as an important component of renin-angiotensin system,plays an important role in the process of cardiac hypertrophy.It has been found that reducing mitochondrial damage,intracellular Ca²⁺overload and oxidative stress can significantly reduce AngII-induced cardiac hypertrophy,but the molecular and cellular mechanisms remain unclear.20-hydroxyeicosatetraenoic acid（20-HETE）is a bioactive lipid mediator produced by arachidonic acid（AA）catalyzed by cytochrome P-450（CYP）enzyme.Current studies have shown that 20-HETE can participate in the regulation of vasoconstriction by stimulating smooth muscle cells contraction,migration and proliferation,as well as activating endothelial cell dysfunction and inflammation.Therefore,20-HETE is considered to be a potent vasoconstrictor and associated with various cardiovascular diseases.Recent research reveals that 20-HETE is also involved in variety of cardiac diseases such as myocardial ischemia-reperfusion injury,myocardial apoptosis and cardiac hypertrophy.Our previous studies have shown that 20-HETE can directly stimulate cardiac hypertrophy,but its exact molecular mechanisms remains to be further elucidated.A large number of researches indicate that 20-HETE can induce intracellular Ca²⁺overload,stimulate reactive oxygen species（ROS）production and cause mitochondrial dysfunction in cardiomyocytes.These actions of 20-HETE may contribute to the cardiac hypertrophy effects of 20-HETE in cardiomyocytes.Interestingly,these effects of 20-HETE are similar to the mechanisms of AngII-induced cardiac hypertrophy,suggesting that Ang II and 20-HETE may share the same intracellular signaling pathway causing cardiac hypertrophy.In addition,AngII has been shown to stimulate arachidonic acid metabolism pathway and induce 20-HETE production in vascular smooth muscle cells and kidneys.Further more,20-HETE is thought to be involved in the vasoconstrictive effect of AngII.However,whether 20-HETE mediates AngII-induced cardiac hypertrophy in H9c2cardiomyocyte by stimulating ROS production,leading to myocardial mitochondrial dysfunction and intracellular Ca²⁺overload has not been investigated previously.Thus,the aims of present study is to explore the role and mechanisms of 20-HETE on AngII-induced cardiac hypertrophy in H9c2 cardiomyocytes,so as to provide theoretical basis for clinical prevention and treatment of AngII-related cardiac hypertrophy.Objectives:1.To observe the role of 20-HETE in AngII-induced cardiac hypertrophy.2.To explore the molecular cellular mechanisms of 20-HETE-mediated AngII-induced cardiac hypertrophy.Methods:H9c2 cardiomyocytes were cultured in vitro and randomly divided into 5 groups（n=6）:control group,AngII group（1μmol/L）,AngII plus HET0016 group（HET0016,10μmol/L）,HET0016 group（10μmol/L）,20-HETE group（100 nmol/L）.After 24 hours of treatment with different drugs,cell surface area was measured after crystal violet staining;total protein content was detected by BCA method;mRNA expression of ANP and BNP,the hypertrophic biomarkers,was detected by qRT-PCR;NADPH oxidase activity was evaluated by cytochrome C reduction method;intracellular ROS level and mitochondrial superoxide production were measured by oxidant sensitive fluorogenic probe,DHE and MitoSOX,respectively;mitochondrial membrane potential was measured by JC-1fluorescent probe;[Ca²⁺]_i in myocardial cells was detected by Flou-3/AM labelled assay using confocal laser scanning microscope;protein expression of CYP4A,CaN and NFAT3was evaluated by Western Blot;production of 20-HETE in myocardial cells was detected by ELISA method.Results:1.Effects of AngII on CYP4A expression and 20-HETE production in H9c2 cardiomyocytesCompared with the control group,protein expression of 20-HETE synthesizing enzyme,CYP4A,was significantly enhanced after treatment of H9c2 cardiomyocytes with Ang II（P<0.05）.Meanwhile,the content of intracellular 20-HETE was increased significantly after treatment with Ang II for 24 hours（P<0.05）.2.Effect of blockade 20-HETE synthesis on AngII-induced cardiac hypertrophy in H9c2cardiomyocytesCompared with the control group,after treating the H9c2 cells for 24h with AngII（1μmol/L）,the surface area of myocardial cells and the content of total intracellular protein are significantly increased and the expression of ANP and BNP,the markers of cardiac hypertrophy,was significantly up-regulated（P<0.05）.However,co-incubation with HET0016（10μmol/L）,a selective inhibitor of the 20-HETE producing enzyme,to reduce the production of 20-HETE significantly inhibited the above effects of AngII（P<0.05）.As a positive control,after treatment of cardiomyocytes with 20-HETE（100nmol/L）alone,myocardial hypertrophy was also significantly promoted,mimicking the action of AngII in H9c2 cardiomyocytes（P<0.05）.3.Effects of AngII and blockade 20-HETE synthesis on ROS production in cardiomyocytesROS plays an important role in AngII-induced cardiac hypertrophy,therefore we first examined the effect of blockade 20-HETE on AngII-induced ROS production in H9c2cardiomyocytes,and found that ROS production in cardiomyocytes of HET0016 plus AngII group was significantly lower than that of AngII group（P<0.05）.In addition,compared with control group,treatment with 20-HETE alone also increased production of ROS in cardiomyocytes（P<0.05）.NADPH oxidase is an important source of ROS production in cardiomyocytes.In this study,the effect of AngII-stimulated NADPH oxidase activity were markedly attenuated by co-treatment with HET0016,which reduce the production of 20-HETE（P<0.05）,and 20-HETE also significantly increased the activity of NADPH oxidase（P<0.05）.4.Effects of AngII and blockade 20-HETE synthesis on mitochondria superoxide production and membrane potential（Δ?m）It has been reported that disruption of myocardial mitochondrial is involved in AngII-induced cardiac hypertrophy.In this study,compared with control group,production of superoxide in mitochondria in AngII group increased significantly,whileΔ?m decreased significantly,both of which were significantly inhibited after co-treatment with HET0016（P<0.05）.In addition,20-HETE could also induce the formation of mitochondrial superoxide and decreaseΔ?m,which is similar to the effect of AngII.5.Effects of AngII and blockade 20-HETE synthesis on[Ca²⁺]_i in cardiomyocytesIt has been reported that persistent increases in intracellular Ca²⁺of cardiomyocytes plays a central role in the pathogenesis of cardiac hypertrophy.Thus,we examined the effect of AngII and HET0016 on[Ca²⁺]_i in H9c2 cardiomyocytes.And the results from this study show that AngII significantly increased,while co-treatment with HET0016 blocked this effect of AngII（P<0.05）.6.Effects of blockade 20-HETE synthesis on AngII-induced protein expression of CaN and NFAT3To further explore the role of 20-HETE on AngII-induced cardiac hypertrophy,we examined the effect of HET0016 on the AngII-induced activation of CaN/NFAT3 signaling pathway.Compared with the control group,the protein expression of CaN and NFAT3 in cardiomyocytes of AngII group was significantly increased（P<0.05）.These effects of AngII were significantly inhibited by co-incubation with HET0016,a 20-HETE synthase inhibitor（P<0.05）.Meanwhile,the treatment of cardiomyocytes with 20-HETE alone did also significantly up-regulated the expression of CaN and NFAT3（P<0.05）.Conclusions:1.20-HETE mediates AngII-induced cardiac hypertrophy by the mechanisms of inducing ROS production and mitochondrial damage.2.20-HETE involves in AngII-induced cardiac hypertrophy by inducing calcium overload to activate CaN/NFAT3 signaling pathway.